PROJECT SUMMARY The epidermis consists of distinct cell layers that work in concert to execute a wide variety of essential functions. Keratinocytes comprise the majority of the cells of the epidermis, and form distinct strata from the basal proliferative layer to the outermost cornified layer. Keratinocytes of all strata secrete a variety of protein factors that have critical roles in the proper function of the epidermis, including structural proteins, antimicrobial peptides, proteases and protease inhibitors, and cell-cell signaling molecules. Despite the recognized role of extracellular proteins in epidermal homeostasis, a comprehensive evaluation of these secreted factors?the keratinocyte ?secretome?? has not been performed. This proposal describes the comprehensive catalog of secreted epidermal proteins we have identified by mass spectrometry. Analysis of the secretome of progenitor and differentiated keratinocytes reveals >100 novel factors whose function in the epidermis is unknown. Studying them one-by-one or through a candidate approach would be a time and resource-intensive effort. Here, we propose to use a CRISPR/Cas9-based genetic screen to systematically identify the functional keratinocyte secreted proteins that impact epidermal renewal and differentiation. This effort would accelerate our understanding of the function of secreted proteins in the skin, and we pave the way to identify new skin biomarkers and therapeutics. 1